Subgenomic replicons of the flavivirus dengue

ABSTRACT

The present invention discloses the construction of dengue virus subgenomic replicons containing large deletions in the structural region (C-preM-E) of the genome, which replicons are useful as vaccines to protect against dengue virus infection.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/656,721, filed Sep. 5, 2003, which is a continuation and claims thebenefit of priority of International Application No. PCT/US02/06962filed Feb. 21, 2002, designating the United States of America andpublished in English, which claims the benefit of priority of U.S.Provisional Application No. 60/274,684 filed Mar. 9, 2001, both of whichare hereby expressly incorporated by reference in their entireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledNIH202-001C1C1_Sequence Listing.TXT, created Feb. 20, 2009, which is 7Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention discloses the construction of dengue virussubgenomic replicons containing large deletions in the structural region(C-preM-E) of the genome, which replicons are useful as vaccines toprotect against dengue virus infection.

BACKGROUND OF THE INVENTION

The mosquito-borne flavivirus, dengue, is estimated to cause in eachyear 100 million cases of dengue fever (DF), 500,000 cases of denguehemorrhagic fever (DHF) and 25,000 deaths, with 2.5 billion people atrisk (Monath, T. P. 1994 PNAS USA 91:2395-2400). Although a successfulvaccine against the prototypical flavivirus, yellow fever (YF) virus,has been in use since the 1930s and vaccines to two other flaviviruses,Japanese encephalitis (JE) virus and tick-borne encephalitis (TBE) virusare currently available, there is as yet no dengue vaccine approved foruse (Cardosa, M. J. 1998 Brit Med Bull 54:395-405).

Dengue virus has a typical flavivirus genome structure, as described inFIG. 2A. The structural proteins, C, prM (M) and E, are involved inpackaging, export and subsequent entry. The non-structural proteins,NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 include an RNA-directed RNApolymerase, and a protease function involved in cleaving certainpositions of the long viral polyprotein which contains all the viralgenes (Chambers, T. J. et al. 1990 Ann Rev Microbiol 44:649-88; Rice, C.M. 1996 In: Fields Virology, 3rd ed. Philadelphia, Pa. Lippincott-RavenPublishers, p. 931-996).

The four serotypes of dengue virus (“1” through “4”) share approximately60%-74% amino acid residue identity with one another in the E gene(Thomas, C. J. et al. 1990 Ann Rev Microbiol 44:649-88) and inducecross-reacting antibodies (Heinz, F. X. 1986 Adv Vims Res 31:103-168).However, neutralizing antibodies to the structural proteins of oneserotype of dengue typically not only fail to provide protection againstother serotypes, but appear to cause the enhanced replication of virusseen in dengue hemorrhagic fever, which is generally seen uponreinfection by dengue virus of a different serotype. Thisantibody-dependent enhancement of infection (ADE), which is believed tobe mediated by enhancement of viral uptake by macrophages (Morens, D. M.1994 Clin Infect Dis 19:500-512) complicates dengue vaccine development,since an inadequate or modified immunogen may contribute to disease,rather than prevent infection (Halstead, S. B. 1988 Science239:476-481).

Two strategies suggest themselves for circumventing the problems causedby cross reacting antibodies against the major structural proteins, prMand E. One strategy is to immunize with multiple strains of dengue virusto elicit high affinity, neutralizing antibodies against the multipledengue serotypes. At least one vaccine to do this (using dengue vaccinecandidates DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK 30/F3, and DEN-4 PDK48)has been in clinical trials (Bhamarapravati, N. and Sutee, Y. 2000Vaccine 18 Suppl 2:4447; Kanesa-thasan, N. et al. 2001 Vaccine19:3179-3188). A second strategy is to induce immunity only to viralproteins other than prM and E. Several studies have shown that thenonstructural glycoprotein NS1 can play an important role in protectionagainst dengue. Mice immunized with purified dengue-2 NS1 proteininjected intramuscularly and boosted after 3 days and two weeks wereprotected from developing lethal dengue encephalitis upon subsequentchallenge with dengue-2 virus (Schlesinger, J. J. et al. 1987 J GenVirol 68:853-857). Similarly, mice immunized with recombinant vacciniavirus expressing authentic NS1 (Falgout, B. et al. 1990 J Virol64:4356-4363) were protected against the development of dengue-4 virusencephalitis when challenged by intracerebral injection. Inoculation ofmice with specific combinations of MAbs directed against dengue-2 NS1(Henchal, E. A. et al. 1988 J Gen Virol 69:2101-2107) also protectsagainst lethal virus encephalitis upon intracerebral dengue-2 challenge.Other nonstructural proteins are also immunogenic and may participate ineliciting protection (Brinton, M. A. et al. 1998 Clin Diagn Virol10:129-39).

SEGUE TO SUMMARY OF THE INVENTION

Towards the goal of devising a “live” vaccine based on onlynon-structural dengue proteins, we have attempted to construct denguevirus genomes from which the pre-M and E genes have been deleted. Uponintroduction into a host's cells, these sub-genomic fragments shouldreplicate intracellularly and support prolonged expression of denguenon-structural proteins without producing the deleted structuralproteins and without forming infectious virions. Sub-genomic repliconsof several positive-strand RNA animal viruses have been reported,particularly yellow fever and Kunjin among the flaviviruses. Thesereplicons, when introduced into host cells, replicate and make viralproteins for over 41 days (Khromykn, A. A. and Westaway, E. D. 1997 JVirol 71:1497-1505), but cannot form infectious virions because theylack critical structural proteins. Effectively delivered to host cellsin vivo, such replicons should efficiently induce immunologic reactionsagainst the expressed proteins remaining in the sub-genomic construct.Herein we describe the successful construction of two dengue virussub-genomic constructs which replicate in LLC-MK2 cells in tissueculture when transfected in as full length RNA. We also report thatexpression of dengue virus proteins from these replicons can besupported by transfection of a DNA-based expression vector containingthe replicon.

SUMMARY OF THE INVENTION

The present invention discloses the construction of dengue virussubgenomic replicons containing large deletions in the structural region(C-preM-E) of the genome, which replicons are useful as vaccines toprotect against dengue virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Representation of ΔE, ΔME, and ΔCME dengue replicons. The ΔEreplicon contains a deletion of the sequence coding for Envelope (E)protein. The ΔME replicon contains a deletion of the sequence coding forpre-Membrane (prM) and Envelope (E) proteins. The ΔCME contains adeletion of the sequence coding for Core (C), pre-Membrane (prM), andEnvelope (E) proteins.

FIG. 2. A, Diagram of dengue virus genome and deletion mutations in thisstudy. B, Sequences at the deletion points of mutants used in thisstudy. ΔME Replicon: (AGTTGT, SEQ ID NO: 1; CGTAACAGCACC, SEQ ID NO: 2;GGTTCT, SEQ ID NO: 3); ΔCME Replicon (AGTTGT, SEQ ID NO: 4;GAGAGAAGCACC, SEQ ID NO: 5; GGTTCT, SEQ ID NO: 6).

FIG. 3. Expression of dengue virus as determined by immunofluorescentstaining with mouse anti-dengue virus-2. Cells were counterstained withEvans Blue, but different filtration systems available on the differentmicroscopes variably blocked visualization of the background of cellsfluorescing red. Transfection efficiencies were generally in the rangeof 0.01% to 1% and in the background of each photograph in this figureare numerous non-fluorescent cells, best visualized on an AppleMacintosh. In the experiments herein, cells were photographed variouslywith either 40× or 60× objectives. A, Dengue-2 wild type virus (48hrs.); B, ΔprM-E (48 hrs.); C, ΔC-prM-E (48 hrs.); D, Dengue-2 wild type(8 days); E, ΔprM-E (8 days); F, ΔC-prM-E (8 days).

FIG. 4. Dengue virus RNA in transfected cells as determined by RT-PCR,normalized to total RNA: Lane 1, λ Hind-Ill molecular weight markers;Lanes 2-4, ΔprM-E; 5-7, ΔE; 2 and 5, 6 hrs.; 3 and 6, 24 hrs.; 4 and 7,48 hrs.

FIG. 5. Expression of dengue virus proteins by DNA-ΔprM-E transfectedinto cells in the form of plasmid DNA.

FIG. 6. Construction of wild type dengue virus and dengue virus repliconvectors used in these studies. The diagram at the top represents thewild type dengue virus genome.

FIG. 7. Expression of green fluorescent protein (GFP) by Δpre-M/E-GFP 48hours post transfection.

FIG. 8. Expression of proteins by Δpre-M/E-gp120 50 hours posttransfection. Left and right frames are two independent fields.Anti-dengue serum was used in these experiments.

FIG. 9. Expression of proteins by Δpre-M/E-gp120 48 hours posttransfection. Left and right frames are two independent fields. Anti-HIVserum was used in these experiments.

FIG. 10. Expression of gp160 by Δpre-M/E-gp160 48 hours posttransfection. Left and right frames are independent fields. Anti-HIVserum was used in these experiments.

FIG. 11. Expression of gp160 by Δpre-M/E-gp160 36 hours posttransfection. Anti-dengue serum was used in these experiments. Left andright panels are independent fields.

FIG. 12. Expression of proteins by Δpre-M/E-gp120 9 days posttransfection. Cells were trypsinized and replated on day 7 posttransfection and harvested for immunofluorescence two days later. Leftand right frames are two independent fields. Anti-dengue serum was usedin these experiments

FIG. 13. Simultaneous expression of HIV and dengue proteins byΔpre-M/E-gp120-transfected cells 4 days post transfection. Left frame:FITC detection of HIV proteins. Right frame: Rhodamine detection ofdengue proteins.

FIG. 14. Simultaneous expression of HIV and dengue proteins byΔpre-M/E-gp120-transfected cells 7 days post transfection. Left frame:FITC detection of HIV proteins. Right frame: Rhodamine detection ofdengue proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention relates to a subgenomic replicon of dengue virus origincomprising a deletion for the sequence coding for C, PreM, and Estructural proteins (ΔCME).

The invention also relates to a subgenomic replicon of dengue virusorigin comprising a deletion for the sequence coding for PreM and Estructural proteins (ΔME).

The invention also relates to a subgenomic replicon of dengue virusorigin comprising a deletion for the sequence coding for E structuralprotein (ΔE).

The invention also relates to a subgenomic replicon of dengue virus type1 origin comprising a deletion for the sequence coding for C, PreM, andE structural proteins (ΔCME).

The invention also relates to a subgenomic replicon of dengue virus type1 origin comprising a deletion for the sequence coding for PreM and Estructural proteins (ΔME).

The invention also relates to a subgenomic replicon of dengue virus type1 origin comprising a deletion for the sequence coding for E structuralprotein (ΔE).

The invention also relates to a subgenomic replicon of dengue virus type2 origin comprising a deletion for the sequence coding for C, PreM, andE structural proteins (ΔCME).

The invention also relates to a subgenomic replicon of dengue virus type2 origin comprising a deletion for the sequence coding for PreM and Estructural proteins (ΔME).

The invention also relates to a subgenomic replicon of dengue virus type2 origin comprising a deletion for the sequence coding for E structuralprotein (ΔE).

The invention also relates to a subgenomic replicon of dengue virus type3 origin comprising a deletion for the sequence coding for C, PreM, andE structural proteins (ΔCME).

The invention also relates to a subgenomic replicon of dengue virus type3 origin comprising a deletion for the sequence coding for PreM and Estructural proteins (ΔME).

The invention also relates to a subgenomic replicon of dengue virus type3 origin comprising a deletion for the sequence coding for E structuralprotein (ΔE).

The invention also relates to a subgenomic replicon of dengue virus type4 origin comprising a deletion for the sequence coding for C, PreM, andE structural proteins (ΔCME).

The invention also relates to a subgenomic replicon of dengue virus type4 origin comprising a deletion for the sequence coding for PreM and Estructural proteins (ΔME).

The invention also relates to a subgenomic replicon of dengue virus type4 origin comprising a deletion for the sequence coding for E structuralprotein (ΔE).

The invention also relates to a subgenomic replicon of dengue virusorigin comprising a deletion for the sequence coding for C, PreM, and Estructural proteins (ΔCME), for PreM and E structural proteins (ΔME), orfor E structural protein (ΔE); and further comprising part or all of the5′UTR; at least about the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, or 175 nucleotides of C protein; at least about the last1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159. 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or 175 nucleotides ofE protein; substantially all of the nonstructural region; and part orall of the 3′UTR.

The invention also relates to a subgenomic replicon of dengue virusorigin comprising a deletion for the sequence coding for C, PreM, and Estructural proteins (ΔCME), for PreM and E structural proteins (ΔME), orfor E structural protein (ΔE), which is adapted to receive at least anucleotide sequence without disrupting its replication capabilities.

The invention also relates to a vaccine comprising a subgenomic repliconof dengue virus origin which comprises a deletion for the sequencecoding for C, PreM, and E structural proteins (ΔCME), for PreM and Estructural proteins (ΔME), or for E structural protein (ΔE), optionallywhich is adapted to receive at least a nucleotide sequence withoutdisrupting its replication capabilities, and a pharmaceuticallyacceptable carrier.

The invention also relates to a therapeutic comprising a subgenomicreplicon of dengue virus origin which comprises a deletion for thesequence coding for C, PreM, and E structural proteins (ΔCME), for PreMand E structural proteins (ΔME), or for E structural protein (ΔE),optionally which is adapted to receive at least a nucleotide sequencewithout disrupting its replication capabilities, and a pharmaceuticallyacceptable carrier.

The invention also relates to a dengue virus like particle comprising asubgenomic replicon of dengue virus origin which comprises a deletionfor the sequence coding for C, PreM, and E structural proteins (ΔCME),for PreM and E structural proteins (ΔME), or for E structural protein(ΔE), optionally which is adapted to receive at least a nucleotidesequence without disrupting its replication capabilities, and structuralproteins of the homologous dengue virus wherein said structural proteinsencapsulate said subgenomic replicon.

The invention also relates to a method of immunization comprisingadministering to an individual in need thereof a subgenomic replicon ofdengue virus origin which comprises a deletion for the sequence codingfor C, PreM, and E structural proteins (ΔCME), for PreM and E structuralproteins (ΔME), or for E structural protein (ΔE), optionally which isadapted to receive at least a nucleotide sequence without disrupting itsreplication capabilities.

The invention also relates to a method of immunization comprisingadministering to an individual in need thereof a dengue virus likeparticle which comprises a subgenomic replicon of dengue virus origincomprising a deletion for the sequence coding for C, PreM, and Estructural proteins (ΔCME), for PreM and E structural proteins (ΔME), orfor E structural protein (ΔE), optionally which is adapted to receive atleast a nucleotide sequence without disrupting its replicationcapabilities, and structural proteins of the homologous dengue viruswherein said structural proteins encapsulate said subgenomic replicon.

The invention also relates to a method of treatment comprisingadministering to an individual in need thereof a subgenomic replicon ofdengue virus origin which comprises a deletion for the sequence codingfor C, PreM, and E structural proteins (ΔCME), for PreM and E structuralproteins (ΔME), or for E structural protein (ΔE), optionally which isadapted to receive at least a nucleotide sequence without disrupting itsreplication capabilities.

The invention also relates to a method of treatment comprisingadministering to an individual in need thereof a dengue virus likeparticle which comprises a subgenomic replicon of dengue virus origincomprising a deletion for the sequence coding for C, PreM, and Estructural proteins (ΔCME), for PreM and E structural proteins (ΔME), orfor E structural protein (ΔE), optionally which is adapted to receive atleast a nucleotide sequence without disrupting its replicationcapabilities, and structural proteins of the homologous dengue viruswherein said structural proteins encapsulate said subgenomic replicon.

Dengue Genome

Dengue viruses are part of the Flavivirus genus in the Flaviviridaefamily. Flaviviruses are single stranded RNA (ssRNA) positive strandviruses which do not have a DNA stage. The flavivirus genome isapproximately 11 kb and consists of a long Open Reading Frame (ORF)flanked by a 5′ Untranslated Region (UTR) (95-132 nt) and a 3′UTR(114-624 nt). The UTRs contain conserved RNA elements and play a role inviral RNA replication. The ORF contains three structural proteins, Core(C), pre-Membrane (prM), Envelope (E), and seven non-structuralproteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

Subgenomic Replicons

Although the present invention describes a means for producing proteins,the term “protein” should be understood to include within its scopeparts of proteins such as peptide and polypeptide sequences.

In use, the replicon is introduced into a host cell where geneexpression and hence protein production take place. Because the vectoris capable of self-replication, multiple copies of the replicon willalso be generated. This leads to an exponential increase in the numberof replicons in the host cell as well as an exponential increase in theamount of protein that is produced.

Optionally, upon introduction of a second vector, containing thestructural genes necessary to produce virus particles, structuralproteins are produced. These proteins encapsulate the replicon thereinforming a “pseudo” recombinant virus that is capable of producingheterologous protein inside another cell. The pseudo-virus cannot,however, replicate to produce new viral particles because the genesnecessary for the production of the structural proteins are not providedin the replicon. Pseudo-virus stock will only be produced whenco-transfection of the replicon and the vector bearing the structuralgenes occurs.

It will be appreciated that any replicon derived from a dengue RNA,which is lacking at least a structural gene, and, optionally, which isadapted to receive at least a nucleotide sequence may be employed in thepresent invention. Preferably, the replicon is derived from dengue types1, 2, 3, or 4 and is adapted to comprise (open language) or consist of(closed language) the following: part or all of the 5′UTR; at leastabout the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or175 nucleotides of C protein (preferably the first 20 to 60nucleotides); at least about the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, or 175 nucleotides of E protein (preferably the last72 nucleotides); substantially all of the nonstructural region; and partor all of the 3′UTR. Replication of a flavivirus genome is dependent onthe genes in the nonstructural region of the genome being present duringtranscription and translation. Preferably any modification made to thenonstructural region should not interfere with the functional activityof the genes within the nonstructural region of the genome.

Optimal dengue replicon design for transfection into eukaryotic cellsmight also include such sequences as: sequences to promote expression ofthe heterologous gene of interest, including appropriate transcriptioninitiation, termination, and enhancer sequences; as well as sequencesthat enhance translation efficiency, such as the Kozak consensussequence; and an internal ribosomal entry site (IRES) of picornaviruses.

Therefore, while the nucleotide sequence may be placed under the controlof dengue regulatory machinery in the replicon, it may alternatively becontrolled by one or more alternate regulatory elements capable ofpromoting expression. Such elements will be well known to those ofordinary skill in this field. In one embodiment of the invention thereplicon contains a eucaryotic promoter sequence (such as a CMVpromoter) upstream of the 5′UTR and a ribozyme sequence (such as a deltavirus ribozyme) followed by a transcription terminator sequencedownstream of the 3′UTR. Transfection of the resulting plasmid DNA incells will ensure production of a dengue replicon RNA transcript withthe authentic 5′-end by cellular RNA polymerase II and with theauthentic 3′-end cleaved by delta virus ribozyme, which is preferred forits efficient replication.

It will be appreciated that the nucleotide sequence inserted into thereplicon may encode part or all of any natural or recombinant proteinexcept for the structural protein sequence into which or in place ofwhich the nucleotide sequence is inserted. For example, the nucleotidesequence may encode a single polypeptide sequence or a plurality ofsequences linked together in such a way that each of the sequencesretains its identity when expressed as an amino acid sequence. Where thenucleotide sequence encodes a plurality of peptides, the peptides shouldbe linked together in such a way that each retains its identity whenexpressed. Such polypeptides may be produced as a fusion protein orengineered in such a manner to result in separate polypeptide or peptidesequences.

Where the vector is used to deliver nucleotide sequences to a host cellto enable host cell expression of immunogenic polypeptides, thenucleotide sequence may encode one or more immunogenic polypeptides inassociation with a range of epitopes which contribute to T-cellactivity. In such circumstances the heterologous nucleotide sequencepreferably encodes epitopes capable of eliciting either a helper T-cellresponse or a cytotoxic T-cell (CTL) response or both.

The replicon described herein may also be engineered to express multiplenucleotide sequences allowing co-expression of several proteins such asa plurality of antigens together with cytokines or otherimmunomodulators to enhance the generation of an immune response. Such areplicon might be particularly useful for example in the production ofvarious proteins at the same time or in gene therapy applications.

By way of example only the nucleotide sequence may encode the DNAsequence of one or more of the following: malarial surface antigens;betagalactosidase; green fluorescence protein; any major antigenic viralantigen, e.g., Haemagglutinin from influenza virus or a humanimmunodeficiency virus (HIV) protein such as HIV gp120 (or gp 160) andHIV gag protein or part thereof; any eukaryotic polypeptide such as, forexample, a mammalian polypeptide such as an enzyme, e.g., chymosin orgastric lipase; an enzyme inhibitor, e.g., tissue inhibitor ofmetalloproteinase (TIMP); a hormone, e.g., growth hormone; a lymphokine,e.g., an interferon; a cytokine, e.g., an interleukin (e.g., IL-2, IL-4,IL-6 etc); a chemokine, e.g., macrophage inflammatory protein-2; aplasminogen activator, e.g., tissue plasminogen activator (tPA) orprourokinase; or a natural, modified or chimeric immunoglobulin or afragment thereof including chimeric immunoglobulins having dual activitysuch as antibody enzyme or antibody-toxin chimeras.

To optimize expression of desired foreign proteins, an autocatalyticpeptide cleavage site is added to the replicon. This construct willallow one to place an autocatalytic peptide cleavage site between twoprotein coding sequences. When the protein is expressed from the nucleicacid construct, the autocatalytic peptide cleavage site willautomatically cleave the desired foreign protein.

The table below compares known autocatalytic peptide cleavage sites:

−16           −1 1      7 Mengo virus, strain M GYFSDLLIHDVETNPGPFTFKPRQ (SEQ ID. NO.: 7) (SEQ ID. NO.: 8) Encephalomyocarditis virus,GYFADLLIHDIETNPG PFMAKPKK strain B (SEQ ID. NO.: 9) (SEQ ID. NO.: 10)Encephalomyocarditis virus, GYFADLLIHDIETNPG PFMFRPRK strain Rueckert(SEQ ID. NO.: 11) (SEQ ID. NO.: 12) Theilers murine DYYRQRLIHDVETNPGPVQSVFQP encephalomyelitis virus, (SEQ ID. NO.: 13) (SEQ ID. NO.: 14)strain Bean Theilers murine DYYKQRLIHDVEMNPG PVQSVFQP encephalomyelitisvirus, (SEQ ID. NO.: 15) (SEQ ID. NO.: 16) strain GDVII Foot-and-mouthdisease NFDLLKLAGDVESNPG PFFFSDVR virus, strain 01 Kaufbeuren (SEQ ID.NO.: 17) (SEQ ID. NO.: 18) Bovine rotavirus type C QIDRILISGDIELNGPPNALVKLN (SEQ ID. NO.: 19) (SEQ ID. NO.: 20) Porcine rotavirus type CQIDRILISGDVELNPG PDPLIRLN (SEQ ID. NO.: 21) (SEQ ID. NO.: 22)

Preferably, the autocatalytic peptide cleavage site is at least 17 aminoacids in length with 16 amino acids extending in the minus direction(NH2 direction) and one residue in the plus direction (COOH direction)relative to the cleavage site. Ryan and Drew, EMBO J 13: 928 (1994)added 3 amino acids (QLL) extending in the minus direction correspondingto the three C-terminal residues of capsid protein 1D aproposfoot-and-mouth disease virus 2A oligopeptide to mediate cleavage of anartificial polyprotein. Other peptide cleavage sites, autocatalytic orotherwise, are contemplated that act as a protease.

The second vector that contains the dengue structural gene(s) should beengineered to prevent recombination with the self-replicating expressionvector. One means for achieving this end is to prepare the second vectorfrom genetic material that is heterologous in origin to the origin ofthe self-replicating expression vector. For example, the second vectormight be prepared from Semliki Forest virus (SFV) as the replicon isprepared from dengue virus.

To optimize expression of the dengue structural genes, the second vectormight include such sequences as: sequences to promote expression of thegenes of interest, including appropriate transcription initiation,termination, and enhancer sequences; as well as sequences that enhancetranslation efficiency, such as the Kozak consensus sequence.Preferably, the second vector contains separate regulatory elementsassociated with each of the different structural genes expressed by thevector. Most preferably, the dengue C gene and the prME genes are placedunder the control of separate regulatory elements in the vector.

The present invention also provides stable cell lines capable ofpersistently producing replicon RNAs. To prepare such cell lines, thedescribed vectors are constructed in selectable form by inserting anIRES-Neo (neomycin transferase) or such cassette into the 3′UTR, the5′UTR, in place of a structural gene, or other.

Host cell lines contemplated to be useful in the method of the inventioninclude any eukaryotic cell lines that can be immortalized, i.e., areviable for multiple passages, (e.g., greater than 50 generations),without significant reduction in growth rate or protein production.Useful cell lines should also be easy to transfect, be capable of stablymaintaining foreign RNA with an unarranged sequence, and have thenecessary cellular components for efficient transcription, translation,post-translation modification, and secretion of the protein. Currentlypreferred cells are those having simple media component requirements,and which can be adapted for suspension culturing. Most preferred aremammalian cell lines that can be adapted to growth in low serum orserum-free medium. Representative host cell lines include BHK (babyhamster kidney), VERO, C6-36, COS, CHO (Chinese hamster ovary), myeloma,HeLa, fibroblast, embryonic and various tissue cells, e.g., kidney,liver, lung and the like. Desirably a cell line is selected from one ofthe following: BHK21 (hamster), SK6 (swine), VERO (monkey), L292(mouse), HeLa (human), HEK (human), 2fTGH cells, HepG2 (human). Usefulcells can be obtained from the American Type Culture Collection (ATCC),Manassas, Va.

With respect to the transfection process used in the practice of theinvention, all means for introducing nucleic acids into a cell arecontemplated including, without limitation, CaPO₄ co-precipitation,electroporation, DEAE-dextran mediated uptake, protoplast fusion,microinjection and lipofusion. Moreover, the invention contemplateseither simultaneous or sequential transfection of the host cell withvectors containing the gene sequences. In one preferred embodiment, hostcells are sequentially transfected with at least two unlinked vectors,one of which contains dengue replicon expressing heterologous gene, andthe other of which contains the structural genes.

The present invention also provides virus like particles containingdengue replicons and a method for producing such particles. It will beappreciated by those skilled in the art that virus like particles thatcontain dengue derived replicons can be used to deliver any nucleotidesequence to a cell. Further, the replicons may be of either DNA or RNAin structure. One particular use for such particles is to delivernucleotide sequences coding for polypeptides that stimulate an immuneresponse. Such particles may be employed as a therapeutic or incircumstances where the nucleotide sequence encodes peptides that arecapable of eliciting a protective immune response so that they may beused as a vaccine. Another use for such particles is to introduce into asubject a nucleotide sequence coding for a protein that is eitherdeficient or is being produced in insufficient amounts in a cell.

The replicon containing dengue virus like particles that containnucleotide coding sequence for immunogenic polypeptide(s) as activeingredients may be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The dengue replicontherapeutic(s) may also be mixed with excipients that arepharmaceutically acceptable and compatible with the repliconencapsulated viral particle. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the therapeutic may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvant which enhance the effectiveness ofthe therapeutic.

The replicon containing dengue virus like particles may beconventionally administered parenterally, by injection, for example,either subcutaneously or intramuscularly. Additional formulations whichare suitable for other modes of administration include suppositoriesand, in some cases, oral formulations. For suppositories, traditionalbinders and carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of virus like particles, preferably 25-70%.

The dengue virus like particles may be formulated into the vaccine asneutral or salt forms. Pharmaceutically acceptable salts include theacid addition salts (formed with free amino groups of the peptide) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids such as acetic, oxalic,tartaric, maleic, and the like. Salts formed with the free carboxylgroups may also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropylamins, trimethylamine, 2-ethylamino ethanol,histidino, procaine, and the like.

The dengue virus like particles may be administered in a mannercompatible with the dosage formulation and in such amount as will beprophylactically and/or therapeutically effective. The dose of viralparticles to be administered depends on the subject to be treated, thetype of nucleotide sequence that is being administered and the type ofexpression efficiency of that sequence and in the case where thenucleotide sequence encodes immunogenic peptide/polypeptides the degreeof protection desired. Precise amounts of active ingredient required tobe administered may depend on the judgment of the practitioner and maybe peculiar to each subject.

The dengue virus like particles may be given to a subject in a singledelivery schedule, or preferably in a multiple delivery schedule. Amultiple delivery schedule is one in which a primary course of deliverymay be with 1-10 separate doses, followed by other doses given atsubsequent time intervals required to maintain and or re-enforce theeffect sought and if needed, a subsequent dose(s) after several months.The delivery regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgment of thepractitioner.

Genetic Immunization and Gene Therapy

In some embodiments, the present invention relates to genetic methods ofeliciting immune responses in an individual which can protect anindividual from dengue infection or combat dengue diseases. According tothe present invention, genetic material that encodes a subgenomicreplicon is directly administered to an individual either in vivo or tothe cells of an individual ex vivo. The genetic material encodes apeptide or protein that shares at least an epitope with a denguenonstructural protein to be targeted. The genetic material is expressedby the individual's cells to form immunogenic target proteins thatelicit an immune response. The resulting immune response is broad based:in addition to a humoral immune response, both arms of the cellularimmune response are elicited. Thus, the immune responses elicited byvaccination methods of the present invention are particularly effectiveto protect against dengue infection or combat diseases associated withdengue.

The immune response elicited by the target protein that is produced byvaccinated cells in an individual is a broad-based immune response whichinvolves B cell and T cell responses including cytotoxic T cell (CTL)responses. The target antigens produced within the cells of the host areprocessed intracellularly: broken down into small peptides, bound byClass I MHC molecules, and expressed on the cell surface. The Class IMHC-target antigen complexes are capable of stimulating CD8+ T-cells,which are phenotypically the killer/suppressor cells. Geneticimmunization according to the present invention is thus capable ofeliciting cytotoxic T-cell (CTL) responses (killer cell responses). Ithas been observed that genetic immunization according to the presentinvention is more likely to elicit CTL responses than other methods ofimmunization.

Genetic immunization according to the present invention elicits aneffective immune response without the use of infective agents orinfective vectors. Vaccination techniques which usually do produce a CTLresponse do so through the use of an infective agent. A complete, broadbased immune response is not generally exhibited in individualsimmunized with killed, inactivated or subunit vaccines. The presentinvention achieves the full complement of immune responses in a safemanner without the risks and problems associated with vaccinations thatuse infectious agents.

According to some embodiments of the present invention, cells aretreated with compounds that facilitate uptake of genetic constructs bythe cells. According to some embodiments of the present invention, cellsare treated with compounds that stimulate cell division and facilitateuptake of genetic constructs. Administration of compounds thatfacilitate uptake of genetic constructs by the cells including cellstimulating compounds results in a more effective immune responseagainst the target protein encoded by the genetic construct.

According to some embodiments of the present invention, the geneticconstruct is administered to an individual using a needleless injectiondevice. According to some embodiments of the present invention, thegenetic construct is simultaneously administered to an individualintradermally, subcutaneously and intramuscularly using a needlelessinjection device. Administration of genetic constructs using needlelessinjection devices is disclosed in the art.

According to the present invention, DNA or RNA that encodes a targetprotein is introduced into the cells of an individual where it isexpressed, thus producing the target protein. The DNA or RNA is linkedto regulatory elements necessary for expression in the cells of theindividual. Regulatory elements for DNA include a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct.

The genetic construct of a genetic vaccine comprises a nucleotidesequence that encodes a target protein operably linked to regulatoryelements needed for gene expression. Accordingly, incorporation of theDNA or RNA molecule into a living cell results in the expression of theDNA or RNA encoding the target protein and thus, production of thetarget protein.

When taken up by a cell, the genetic construct which includes thenucleotide sequence encoding the target protein operably linked to theregulatory elements may remain present in the cell as a functioningextrachromosomal molecule or it may integrate into the cell'schromosomal DNA. DNA may be introduced into cells where it remains asseparate genetic material in the form of a plasmid. Alternatively,linear DNA which can integrate into the chromosome may be introducedinto the cell. When introducing DNA into the cell, reagents whichpromote DNA integration into chromosomes may be added. DNA sequenceswhich are useful to promote integration may also be included in the DNAmolecule. Since integration into the chromosomal DNA necessarilyrequires manipulation of the chromosome, it is preferred to maintain theDNA construct as a replicating or non-replicating extrachromosomalmolecule. This reduces the risk of damaging the cell by splicing intothe chromosome without affecting the effectiveness of the vaccine.Alternatively, RNA may be administered to the cell. It is alsocontemplated to provide the genetic construct as a linear minichromosomeincluding a centromere, telomeres and an origin of replication.

The necessary elements of a genetic construct of a genetic vaccineinclude a nucleotide sequence that encodes a target protein and theregulatory elements necessary for expression of that sequence in thecells of the vaccinated individual. The regulatory elements are operablylinked to the DNA sequence that encodes the target protein to enableexpression.

The molecule that encodes a target protein is a protein-encodingmolecule which is translated into protein. Such molecules include DNA orRNA which comprise a nucleotide sequence that encodes the targetprotein. These molecules may be cDNA, genomic DNA, synthesized DNA or ahybrid thereof or an RNA molecule such as mRNA. Accordingly, as usedherein, the terms “genetic construct” and “nucleotide sequence” aremeant to refer to both DNA and RNA molecules.

The regulatory elements necessary for gene expression of a DNA moleculeinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression. It is necessary that these elements be operable in thevaccinated individual. Moreover, it is necessary that these elements beoperably linked to the nucleotide sequence that encodes the targetprotein such that the nucleotide sequence can be expressed in the cellsof a vaccinated individual and thus the target protein can be produced.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the target protein. However, it isnecessary that these elements are functional in the vaccinatedindividual.

Similarly, promoters and polyadenylation signals used must be functionalwithin the cells of the vaccinated individual.

In order to be a functional genetic construct, the regulatory elementsmust be operably linked to the nucleotide sequence that encodes thetarget protein. Accordingly, it is necessary for the initiation andtermination codons to be in frame with the coding sequence.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the vaccinatedcells. Moreover, codons may be selected which are most efficientlytranscribed in the vaccinated cell. One having ordinary skill in the artcan produce DNA constructs which are functional in vaccinated cells.

In order to test expression, genetic constructs can be tested forexpression levels in vitro using tissue culture of cells of the sametype as those to be vaccinated. For example, if the genetic vaccine isto be administered into human muscle cells, muscle cells grown inculture such as solid muscle tumors cells of rhabdomyosarcoma may beused as an in vitro model to measure expression level.

According to the invention, the genetic vaccine may be administereddirectly into the individual to be immunized or ex vivo into removedcells of the individual which are reimplanted after administration. Byeither route, the genetic material is introduced into cells which arepresent in the body of the individual. Routes of administration include,but are not limited to, intramuscular, intraperitoneal, intradermal,subcutaneous, intravenous, intraarterially, intraoccularly and oral aswell as transdermally or by inhalation or suppository. Preferred routesof administration include intramuscular, intraperitoneal, intradermaland subcutaneous injection. Genetic constructs may be administered bymeans including, but not limited to, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gene guns”.Alternatively, the genetic vaccine may be introduced by various meansinto cells that are removed from the individual. Such means include, forexample, ex vivo transfection, electroporation, microinjection andmicroprojectile bombardment. After the genetic construct is taken up bythe cells, they are reimplanted into the individual. It is contemplatedthat otherwise non-immunogenic cells that have genetic constructsincorporated therein can be implanted into the individual even if thevaccinated cells were originally taken from another individual.

The genetic vaccines according to the present invention comprise about 1nanogram to about 1000 micrograms of DNA. In some preferred embodiments,the vaccines contain about 10 nanograms to about 800 micrograms of DNA.In some preferred embodiments, the vaccines contain about 0.1 to about500 micrograms of DNA. In some preferred embodiments, the vaccinescontain about 1 to about 350 micrograms of DNA. In some preferredembodiments, the vaccines contain about 25 to about 250 micrograms ofDNA. In some preferred embodiments, the vaccines contain about 100micrograms DNA.

The genetic vaccines according to the present invention are formulatedaccording to the mode of administration to be used. One having ordinaryskill in the art can readily formulate a genetic vaccine that comprisesa genetic construct. In cases where intramuscular injection is thechosen mode of administration, an isotonic formulation is preferablyused. Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are preferred. Stabilizersinclude gelatin and albumin. In some embodiments, a vaso-constrictionagent is added to the formulation. The pharmaceutical preparationsaccording to the present invention are provided sterile and pyrogenfree.

Genetic constructs may optionally be formulated with one or moreresponse enhancing agents such as: compounds which enhance transfection,i.e., transfecting agents; compounds which stimulate cell division,i.e., replication agents; compounds which stimulate immune cellmigration to the site of administration, i.e., inflammatory agents;compounds which enhance an immune response, i.e., adjuvants or compoundshaving two or more of these activities.

In a preferred embodiment, bupivacaine, a well known and commerciallyavailable pharmaceutical compound, is administered prior to,simultaneously with or subsequent to the genetic construct. Bupivacaineand the genetic construct may be formulated in the same composition.Bupivacaine is particularly useful as a cell stimulating agent in viewof its many properties and activities when administered to tissue.Bupivacaine promotes and facilitates the uptake of genetic material bythe cell. As such, it is a transfecting agent. Administration of geneticconstructs in conjunction with bupivacaine facilitates entry of thegenetic constructs into cells. Bupivacaine is believed to disrupt orotherwise render the cell membrane more permeable. Cell division andreplication is stimulated by bupivacaine. Accordingly, bupivacaine actsas a replicating agent. Administration of bupivacaine also irritates anddamages the tissue. As such, it acts as an inflammatory agent whichelicits migration and chemotaxis of immune cells to the site ofadministration. In addition to the cells normally present at the site ofadministration, the cells of the immune system which migrate to the sitein response to the inflammatory agent can come into contact with theadministered genetic material and the bupivacaine. Bupivacaine, actingas a transfection agent, is available to promote uptake of geneticmaterial by such cells of the immune system as well.

Bupivacaine is related chemically and pharmacologically to the aminoacyllocal anesthetics. It is a homologue of mepivacaine and related tolidocaine. Bupivacaine renders muscle tissue voltage sensitive to sodiumchallenge and effects ion concentration within the cells. A completedescription of bupivacaine's pharmacological activities can be found inRitchie, J. M. and N. M. Greene, The Pharmacological Basis ofTherapeutics, Eds.: Gilman, A. G. et al, 8th Edition, Chapter 15:3111.Bupivacaine and compounds that display a functional similarity tobupivacaine are preferred in the method of the present invention.

Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide,1-butyl-N-(2,6-dimethylphenyl)monohydrochloride, monohydrate and iswidely available commercially for pharmaceutical uses from many sourcesincluding from Astra Pharmaceutical Products Inc. (Westboro, Mass.) andSanofi Winthrop Pharmaceuticals (New York, N.Y.), Eastman Kodak(Rochester, N.Y.). Bupivacaine is commercially formulated with andwithout methylparaben and with or without epinephrine. Any suchformulation may be used. It is commercially available for pharmaceuticaluse in concentration of 0.25%, 0.5% and 0.75% which may be used on theinvention. Alternative concentrations which elicit desirable effects maybe prepared if desired.

Other contemplated response enhancing agents which may functiontransfecting agents and/or replicating agents and/or inflammatory agentsand which may be co-adminstered with bupivacaine and similar actingcompounds include lectins, growth factors, cytokines and lymphokinessuch as alpha-interferon, gamma-interferon, platelet derived growthfactor (PDGF), gCSF, gMCSF, TNF, epidermal growth factor (EGF), IL-1,IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12 as well as collagenase,fibroblast growth factor, estrogen, dexamethasone, saponins, surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freund'sincomplete adjuvant, LPS analog including monophosphoryl Lipid A (MPL),muramyl peptides, quinone analogs and vesicles such as squalene andsqualane, hyaluronic acid and hyaluronidase may also be usedadministered in conjunction with the genetic construct. In someembodiments, combinations of these agents are administered inconjunction with bupivicaine and the genetic construct. For example,bupivacaine and either hyaluronic acid or hyaluronidase areco-administered with a genetic construct.

In some embodiments of the invention, the genetic construct is injectedwith a needleless injection device. The needleless injection devices areparticularly useful for simultaneous administration of the materialintramuscularly, intradermally and subcutaneously.

In some embodiments of the invention, the genetic construct isadministered with a response enhancing agent by means of amicroprojectile particle bombardment procedure as taught by Sanford etal. in U.S. Pat. No. 4,945,050 issued Jul. 31, 1990.

In some embodiments of the invention, the genetic construct isadministered as part of a liposome complex with a response enhancingagent.

In some embodiments of the invention, the individual is subject to asingle vaccination to produce a full, broad immune response. In someembodiments of the invention, the individual is subject to a series ofvaccinations to produce a full, broad immune response. According to someembodiments of the invention, at least two and preferably four to fiveinjections are given over a period of time. The period of time betweeninjections may include from 24 hours apart to two weeks or longerbetween injections, preferably one week apart. Alternatively, at leasttwo and up to four separate injections are given simultaneously atdifferent sites.

While this disclosure generally discusses immunization in the context ofprophylactic methods of protection, the term “immunizing” is meant torefer to both prophylactic and therapeutic methods. Thus, a method ofimmunizing includes both methods of protecting an individual from denguechallenge as well as methods of treating an individual suffering fromdengue disease. Accordingly, the present invention may be used as avaccine for prophylactic protection or in a therapeutic manner; that is,as immunotherapeutic methods and preparations.

Other aspects of the invention include the use of genetic constructs inmethods of introducing therapeutic genes into cells of an individual.Thus, one aspect of the present invention relates to gene therapy; thatis, to methods of introducing nucleic acid molecules that encodetherapeutic proteins into the cells of an individual. The administrationprotocols and genetic constructs useful in gene therapy applications arethe same as those described above for genetic immunization except thegenetic constructs include nucleotide sequences that encode proteinswhose presence in the individual will eliminate a deficiency in theindividual and/or whose presence will provide a therapeutic effect onthe individual.

Construction of Dengue Replicons and Optional Encapsidation

Nucleotide sequences encoding the structural and nonstructural proteinsof all four dengue types have been reported. Type I: Fu et al. 1992Virology 188:953; Type II: Gualano et al. 1998 J Gen Virology 79:437;Type III: Osatomi et al. 1990 Virology 176:643; Type IV: Zhao et al.1986 Virology 155:77; Mackow et al. 1987 Virology 159:217.

Several dengue subgenomic replicons containing large deletions in thestructural region (C-prM-E) can be constructed. Referring to FIG. 1,representative are ΔE, ΔME, and ΔCME dengue replicons. The ΔE repliconcontains a deletion of the sequence coding for Envelope (E) protein. TheΔME replicon contains a deletion of the sequence coding for pre-Membrane(prM) and Envelope (E) proteins. The ΔCME contains a deletion of thesequence coding for Core (C), pre-Membrane (prM), and Envelope (E)proteins.

All deletion constructs may be prepared from cDNA clones used in theconstruction of plasmid dengue for generation of infectious dengue RNAby PCR-directed technology, using appropriate primers and conventionalcloning. ΔC, ΔME, and ΔCME, and derivatives, are prepared to comprise(open language) or consist of (closed language) the following: part orall of the 5′UTR; at least about the first 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, or 175 nucleotides of C protein; at least aboutthe last 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159. 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or 175nucleotides of E protein; substantially all of the nonstructural region;and part or all of the 3′UTR.

Evidence of replication of these RNAs after introduction into host cellsis sought by immunofluorescence analysis of cells with antibodies todengue proteins or by radioimmunoprecipitation analysis of lysates withantibodies to dengue proteins. No apparent cytopathic effect is observedin the great majority of the antibody positive cells at any timeposttransfection. Reverse transcription PCR and Northern blot analysisconfirm accumulation of dengue-specific RNA in cells transfected withΔC, ΔME, and ΔCME, and derivatives.

A minimal sequence in core protein, or envelope protein, required forRNA replication is defined. Replicon constructs containing sequencescoding for only the first amino acids of core protein, or only the lastamino acids of envelope protein, are prepared. Deletions in the C codingsequence, or E coding sequence, are monitored for translation efficiencyand replication activity. Translation efficiency may be unaffected.Replication activity is compared. Immunofluorescence results andNorthern blot analysis may identify the minimal sequence required forRNA replication.

The effect of deletions in the 5′UTR and 3′UTR on RNA replication isdetermined. Replicon constructs with deletions in the 5′UTR or 3′UTR areprepared. Immunofluorescence results and Northern blot analysis mayidentify the 5′UTR and 3′UTR sequence(s) that may be removed withoutdeleterious effect on RNA replication.

Multi-cistronic dengue replicon RNAs expressing heterologous genes areprepared. As a first step, an expression cassette is prepared forinsertion into the 3′UTR, the 5′UTR, in place of a structural gene, orother. Initially, the IRES from EMCV is linked to a reporter gene tocreate an IRES-reporter gene cassette. The IRES from EMCV ensurescap-independent internal initiation of translation of the reporter geneto which it is linked. For example, an IRES-CAT gene cassette is cloned.The expression cassette is inserted in the plus orientation. Synthesisof plus strands of replicon RNA during replication is monitored viaexpression of the CAT gene.

To select for cells persistently expressing dengue replicons, the CATgene is replaced by the Neo gene. The low percentage ofreplicon-expressing cells is strikingly enriched by selection duringgrowth in the presence of the antibiotic G418. No apparent changes inthe morphology of surviving positive cells is observed.

Dengue virus replicon RNA is encapsidated by a procedure involving twosimultaneous or consecutive introductions into host cells, first withdengue virus replicon ΔC, ΔME, or ΔCME RNA, and about 24-hours laterwith a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressingdengue virus structural proteins. The presence of dengue virus repliconRNA in encapsidated particles is demonstrated by its amplification andexpression in host cells, detected by Northern blotting withdengue-specific probes and by immunofluorescence analysis withantibodies to dengue proteins. Infectious particles are pelleted byultracentrifugation of the culture fluid from cells into which areintroduced dengue virus replicon ΔC, ΔME, or ΔCME RNA, and recombinantSemliki Forest virus (SFV) replicon RNA(s) expressing dengue virusstructural proteins. The particles are neutralized by preincubation withantibodies to dengue E protein. Radioimmunoprecipitation analysis withanti-E antibodies of the culture fluid of the doubly transfected cellsshows the presence of C, preM, and E proteins in the immunoprecipitatedparticles. Reverse transcription PCR shows that the immunoprecipitatedparticles also contain dengue specific RNA. The encapsidated repliconparticles sediment about the same as dengue virions in a 5 to 25%sucrose density gradient and are uniformly spherical, with a diameterthat compares favorably with an approximately 50 nm diameter for denguevirions.

Example 1

As part of a program to develop a dengue virus vaccine which avoids thedeleterious effects of antibody dependent enhancement (ADE) of infectionmediated by antibodies to dengue virus structural proteins, weinvestigated the possibility of designing dengue vaccines based onnon-structural proteins.

Our results indicate that dengue constructs which lack major structuralproteins replicate intracellularly in tissue culture. These repliconsare capable of prolonged expression of dengue virus non-structuralproteins for at least seven days in culture.

Our conclusions indicate that dengue virus genomes lacking majorstructural proteins can, like other flaviviruses, replicateintracellularly and express virus non-structural proteins with minimaltoxicity to host cells. These findings pave the way for the developmentof dengue virus replicons as a form of live, attenuated virus vaccine.

Development of Dengue Virus Type 2 Replicons Capable of ProlongedExpression in Host Cells

Immunofluorescent analysis of cell cultures 48 hrs. post transfectiondemonstrates efficient expression of dengue virus proteins from wildtype dengue virus as well as from both the ΔprM-E and ΔC-prM-E mutants(see FIG. 2 and FIGS. 3A, 3B and 3C). By this time point, the wild typevirus has had a limited opportunity to be transmitted in secondaryrounds of infection. Interestingly, pairs of fluorescent cells wereoften seen, suggesting cells continued to replicate after beingsuccessfully transfected by replication competent dengue replicons. Thisis particularly evident in FIGS. 3B and 3C.

By 8 days post transfection, wild type dengue virus expression is morewidespread throughout the culture than it was 48 hrs. post transfection,presumably because it was able to undergo multiple rounds of replicationand transmission (FIG. 3D). Cell cultures transfected with ΔprM-E (FIG.3E) or ΔC-prM-E (FIG. 3F) still have cells efficiently expressing dengueproteins at 8 days, but they are more rare than they were at 48 hrs.post transfection. This is consistent with the inability of thesemutants to make infectious virions and suggests that the viral proteinsremaining in the dengue replicons may moderately retard cell growth andreplication. However, in the experiments presented herein, cells weretrypsinized and replated on day 7 post transfection, so they may nothave had sufficient time to recover and replicate prior to harvest forimmunofluorescence on day 8.

No fluorescent cells were ever seen at either 48 hrs. or 8 days posttransfection with dengue deletion mutant ΔE, from which most of the Egene has been deleted. However, negative results are hard to interpretin this system.

The prolonged expression of dengue virus proteins by the ΔprM-E andΔC-prM-E replicons presumably is dependent on the ability of thesesub-genomic fragments to replicate. Consistent with this presumption,ΔprM-E RNA was seen to sequentially increase in cultures over at leastthe first 48 hrs. post transfection (FIG. 4), whereas no dengue virusRNA was seen over this time period after transfection with ΔE RNA. RNAfrom both of these constructs is undetectable at 6 hrs. posttransfection, suggesting that most of the transfecting RNA is rapidlydegraded.

When the Δ-prM-E replicon is placed under the control of acytomegalovirus (CMVB) promoter, it is still capable of active denguevirus protein expression when transfected into cells in the form ofplasmid DNA (see FIG. 5).

The expression of dengue virus RNA and proteins in cultures transfectedwith the Δ-prM-E and ΔC-prM-E mutants is consistent with replication ofthe viral sub-genomes in these host cells. This is consistent withsimilar replicons constructed from Kunjin virus (Khromykn, A. A. andWestaway, E. D. 1997 J Virol 71:1497-1505). Most encouraging, however,is the finding of viral protein expression at long time points (8 days)subsequent to transfection with the ΔprM-E and ΔC-prM-E replicons in theabsence of selection. Previous experiments with other flavivirusreplicons have demonstrated expression for as long as 41 days posttransfection (Khromykn, A. A. and Westaway, E. D. 1997 J Virol71:1497-1505), but these replicons expressed neomycin and were grownunder selective pressure. The longest, previously reported time for purereplicon expression in the absence of selection was just 72 hours(Khromykn, A. A. and Westaway, E. D. 1997 J Virol 71:1497-1505). We havenot yet searched for continued expression beyond 8 days posttransfection, but we have no reason to believe that is not readilyachievable.

In order for the dengue replicons reported herein to be of immunologicvalue, they need to be expressible in a convenient form and (weanticipate) they need to produce NS1 protein. Formally, the datapresented herein do not directly prove NS1 production, because theantisera used detect multiple viral structural and nonstructuralproteins. Attempts to visualize dengue replicon protein synthesis byWestern blotting were unsuccessful in our hands, presumably because ofthe comparatively low transfection efficiencies achieved. However, thepreviously demonstrated dependence of dengue virus replication on NS1production (Lindenbach, B. D. and Rice, C. M. 1997 J Virol 71:9608-9617)and the fact that dengue virus RNA levels increase with time aftertransfection in the cultures used herein (implying active dengue RNAreplication) together strongly imply the production of significantquantities of NS1 protein by these replicons. As for a suitable form ofdelivery, the ability of pDNA-ΔprM-E to make high levels of dengueproteins after transfection into cells in the form of DNA suggests thepossibility of using DNA transfection to achieve immunization (Beard, C.et al. 1999 J Biot 73:243-249).

In conclusion, we have demonstrated the prolonged expression of denguevirus proteins from sub-genomic dengue RNA fragments, lacking majorstructural genes, transfected into tissue culture cells. This prolongedexpression is associated with detectable increases in dengue RNA in thetransfected tissue cultures, implying that the sub-genomic fragments arereplicating, and implying the synthesis of NS1 protein and other viralnon-structural proteins known to be required for viral genomicreplication. The ability to express these sub-genomic dengue repliconsfrom transfected DNA offers the possibility of using DNA-based, denguereplicon vaccines. Other delivery methods are contemplated, includingthe development of packaging cell lines.

Culturing of Dengue Virus

Dengue virus strains DEN1/WP and DEN2/NGC, kindly provided by Dr. LewisMarkoff (Polo, S. et al. 1997 J Virol 71:536-674; Pur, B. et al. 2000Virus Genes 20:57-63), were passaged in monkey LLC-MK2 cells at 37° C.in a humidified incubator under 5% CO₂, using Medium 199 plus 10% fetalbovine serum (FBS) and 50 μg of Gentamicin per ml. The cells weretrypsinized a day before virus infection and plated to reachapproximately 80% confluence on the day of infection. Infections weretypically at an MOI of 0.01 PFU/cell in Medium 199 plus 2% FBS.

In Vitro Mutagenesis

DNA fragments used for spanning the desired deletions (see FIG. 2) weresynthesized by polymerase chain reaction (PCR) from two shortoverlapping primers. For the ΔCME replicon, the 5′ primer was 5′ATCATTATGCTGATTCCAACAGTGATGGCG TTCCATTTAACCACACGTAACAGCACCTCACTGTCTGTG3′(SEQ ID NO:23). For the ΔprME replicon, the 5′ primer was5′ACAGCTGTCGCTCCTTCAATGACAATGCGTTGCATAGGAATATCAAATAGAAGCACCTCACTGTCTGTG3′ (SEQ ID. NO.: 24). For bothmutants, the 3′ primer was 5′ATACAGCGTCACGACTCCCACCAATACTAGTGACACAGACAGTGAGGTGCT3′ (SEQ ID. NO.: 25).

PDNA-ΔprM-E was constructed by placing the ΔprM-E replicon RNA under thetranscriptional control of a CMV promoter and placing it upstream of aHepatitis Delta Virus (HDV) self cleaving ribozyme.

Dengue-2 virus cDNA cloned in the yeast shuttle vector pBR424,linearized by excision of a short BamHI fragment was transfected intocompetent (Spencer, F. et al. 1993 Methods Companion Methods Enzymol5:161-175) S. cerevisiae YPH857, kindly provided by Barry Falgout(CBER/FDA), along with the appropriate PCR fragment spanning the desireddeletion. Yeast colonies which grew on tryptophan minus platesrepresented vectors which had recircularized by homologous recombinationwith these PCR fragments (Spencer, F. et al. 1993 Methods CompanionMethods Enzymol 5:161-175). DNA from these colonies was transformed intoE. coli Stbl 2 cells (Life Technologies, Inc.) to make sufficientquantities of dengue recombinant, genomic-length DNAs forcharacterization and analysis.

Expression of Virus and Replicons in Cells

The full length virus and replicon cDNA plasmids isolated from Stbl 2cells were linearized with SacI, purified by Qiagen chromatography, andeluted by RNase-free water in preparation for transcription. Thetranscription reaction mixtures contained 1 μg of linearized DNA; 0.5 mM(each) ATP, CTP, and UTP; 0.1 mM GTP; 0.5 mM cap analog (NEBL); 10 mMDTT; 40 U of RNasin (Promega); 30 U of SP6 RNA polymerase; and 1×SP6 RNApolymerase buffer (Promega) in a volume of 30 μl. The reaction mixtureswere incubated at 40° C. for 2 hrs. Aliquots (12.5 μl) of the reactionmixtures, containing full length viral RNA, were used to transfectapproximately 2×10⁶ monkey LLC-MK2 cells in phosphate-buffered saline(PBS) by electroporation in a 0.4 cm gap electroporation cuvette. Eachcuvette was pulsed at 200 V, 950 μF using a BioRad Genepulselectroporator. The cells were then resuspended in growth medium andplated on the appropriate tissue culture dish. Plasmid DNA-ΔprM-E wastransfected into cells by electroporation using identical conditions.

After electroporation, cells were either plated directly on multiwellplates for harvest at short time periods (typically 48 hrs. or less) oron tissue culture dishes for trypsinization and seeding onto multiwellplates on the day before final harvest for longer time periods(typically 8 days post transfection).

Immuno-Histochemical Methods

Cells growing on chamber slides were rinsed in room-temperature PBS andthen fixed in cold acetone for 10 min at −20° C. After being air dried,each chamber was covered with 50 μl of a 1:50 dilution of DEN2-specifichyperimmune mouse ascitic fluid (HMAF, American Type Culture Collection)in PBS plus 2% normal goat serum. Samples were incubated at roomtemperature for 1 h in a humidified atmosphere and then rinsed twice inPBS. Samples were similarly incubated with a 1:100 dilution offluorescein isothiocyanate-labeled goat anti-mouse antibodies (LifeTechnologies) and rinsed twice in PBS. Cells in some experiments werecounterstained with 0.02% Evans Blue.

Example 2

Despite tremendous progress in developing anti-retroviral drugs tocombat HIV, there remains a need for an effective HIV vaccine. This needis particularly pressing in third world countries, where demographicsand economics make drug therapy difficult to deliver. Although HIVinfection elicits neutralizing antibodies and a cellular immune responseagainst the virus (reviewed in Nathanson, N. and Mathieson, B. J. 2000 JInfect Dis 182:579-589; and Cho, M. W. 2000 Adv Pharmacol 49:263-314)and there exist “exposed uninfected” (EU) individuals that appear tohave acquired resistance to infection by HIV (Clerici, M. et al. 1992 JInfect Dis 165:1012-1019; Kaul, R. et al. In: Program and abstracts ofthe 7th Conference on Retroviruses and Opportunistic Infections (SanFrancisco) San Francisco: Foundation for Retrovirology and Human Health,2000, 168), the hallmark of HIV infection is the almost universalinability of humans to mount an immune response that can prevent theeventual development of AIDS.

An effective vaccine will require not only the design of effectiveimmunogens, but also the design of optimized protocols of immunogendelivery. As a live, attenuated vaccine for HIV is considered difficultto test and dangerous to implement (Nathanson, N. and Mathieson, B. J.2000 J Infect Dis 182:579-589; Cho, M. W. 2000 Adv Pharmacol 49:263-314;Baba, T. W. et al. 1999 Nat Med 5:194-203; Baba, T. W. et al. 1995Science 267:1820-1825; Bogers, W. M. et al. 1995 AIDS 9:F13-F18;Greenough, T. C. et al. 1999 N Engl J Med 340:236-237; Berkhout, B. etal. 1999 J Virol 73:1138-1145), various alternatives to HIV could beconsidered as potential “live” vectors for HIV immunogens, includingenteric bacteria, poxviruses (vaccinia and canarypox), small RNA viruses(e.g. poliovirus and Semliki Forest virus), Rhabdoviruses (e.g.vesicular stomatitis virus), DNA viruses (e.g. adenovirus andadeno-associated viruses) and even naked DNA to achieve expression inliving host cells (Cho, M. W. 2000 Adv Pharmacol 49:263-314; Rose, N. F.et al. 2001 Cell 106:539-549).

Dengue possesses several advantages which favor its choice as a vectorfor HIV immunogens. As a flavivirus, it replicates entirely in thecytoplasm through RNA directed RNA polymerization and is incapable ofintegrating into the host genome. Flavivirus replicons can replicateinside cells and achieve prolonged expression of high levels of virallyencoded proteins with minimal toxicity (Khromykn, A. A. and Westaway, E.D. 1997 J Virol 71(2):1497-1505) and are unable to recombine or mutateto produce infectious HIV particles. Finally, by eliciting an immunereaction against the dengue non-structural proteins remaining inreplicons, dengue virus replicons may induce a protective immunityagainst dengue which would not predispose vaccinated individuals to DHF.Properly administered, dengue virus replicons expressing HIV epitopesmight thus serve as dual vaccines, conferring protection against denguevirus as well as HIV.

The challenges in developing a safe and effective HIV vaccine are manyand varied. Choice of immunogen is clearly problematic. Criticalepitopes may be masked by glycosyl groups and/or tertiary structure(Reitter, J. et al. 1998 Nat Med 4:679-684; Chan, D.C. and Kim, P. S.1998 Cell 93:681-684; LaCasse, R. A. et al. 1999 Science 283:357-362)and (Cooney, E. L. et al. 1991 Lancet 337:567-572). The extensivegenetic variability of HIV complicates immunogen choice and the highrate of mutation increases the likelihood of the rapid development ofresistance. Furthermore, the method of immunogen delivery (e.g.,purified subunits or inactivated virus vs. various forms of “live”expression) can determine the relative nature and extent of humoral andcell mediated immunologic responses. Priming with various types of“live” expression followed by boosting with purified subunits iscurrently favored as a method to obtain stronger immunologic responsesthan either method alone (reviewed in Nathanson, N. and Mathieson, B. J.2000 J Infect Dis 182:579-589; and Cho, M. W. 2000 Adv Pharmacol49:263-314). Previously acquired immunity to a viral vector such asvaccinia may influence its efficacy in inducing immunity againstheterologous proteins being delivered (Cooney, E. L. et al. 1991 Lancet337:567-572; Cooney, E. L. et al. 1993 PNAS USA 90:1882-1886; Graham, B.S. et al. 1992 J Infect Dis 166:244-52; McElrath, M. J. et al. 1994 JInfect Dis 169:41-47) and it may be wise to provide physicians with HIVvaccines based on a variety of vectors to handle a variety of clinicalsituations.

The use of live dengue as a vaccine or as a vector for heterologousimmunogens has historically been considered problematic because of thepathologies associated with dengue infection. Although dengue fever (DF)is usually self limited, dengue hemorrhagic fever (DHF) is considerablydebilitating and frequently fatal (Monath, T. P. 1994 PNAS USA91:2395-2400). However, DHF is unlikely to result from or be promoted bythe vectors reported herein. The enhanced replication of virus seen indengue hemorrhagic fever is generally seen upon reinfection by denguevirus of a serotype different from previous infections and is believedto be mediated by antibodies against viral structural proteins: socalled antibody dependent enhancement of infection, or ADE. These crossreacting antibodies actually promote viral uptake by macrophages(Morens, D. M. 1994 Clin Infect Dis 19:500-512; Halstead, S. B. 1988Science 239:476-481). The main challenge in using live dengue in humansis thus avoiding the development of antibody dependent enhancement (ADE)of infection by antibodies against the pre-M and E proteins of onedengue strain which weakly cross react with the pre-M and E of a secondinfecting dengue strain. Since the replicons reported herein lack themajor viral structural protein genes, they are not only incapable ofsustaining a spreading infection but also are incapable of elicitingantibodies against the missing structural proteins. They should neitherinduce DF nor promote DHF.

Dengue virus has a typical flavivirus genome structure, as described inFIG. 6. The structural proteins, C, pre-M (M) and E, are involved inpackaging, export and subsequent entry. The non-structural proteins,NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 include an RNA-directed RNApolymerase and a protease function involved in cleaving certainpositions of the long viral polyprotein which contains all the viralgenes (Chambers, T. J. et al. 1990 Ann Rev Microbiol 44:649-88; Rice, C.M In: Fields Virology, 3rd ed. Philadelphia, Pa. Lippincott-RavenPublishers, 1996 p. 931-996). The four serotypes of dengue virus (“1”through “4”) share approximately 60%-74% amino acid residue identitywith one another in the E gene (Thomas, C. J. et al. 1990 Annu RevMicrobiol 44:649-88) and induce cross-reacting antibodies (Heinz, F. X.1986 Adv Virus Res 31:103-168).

Two strategies suggest themselves for circumventing the problem of ADEfrom dengue vaccination. One strategy is to immunize with multiplestrains of dengue virus to elicit high affinity, neutralizing antibodiesagainst the multiple dengue serotypes. At least one vaccine to do this(using dengue vaccine candidates, DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK30/F3, and DEN-4 PDK48) has been in clinical trials (Bhamarapravati, N.and Sutee, Y. 2000 Vaccine 18 Suppl 2:44-47; Kanesa-thasan, N. et al.2001 Vaccine 19:3179-3188). A second strategy is to induce immunity onlyto viral proteins other than pre-M and E. Several studies have shownthat the nonstructural glycoprotein NS1 can play an important role inprotection against dengue. Mice immunized with purified dengue-2 NS1protein injected intramuscularly and boosted after 3 days and two weekswere protected from developing lethal dengue encephalitis uponsubsequent challenge with dengue-2 virus (Schlesinger, J. J. et al. 1987J Gen Virol 68:853-857). Similarly, mice immunized with recombinantvaccinia virus expressing authentic NS1 (Falgout, B. et al. 1990 J Virol64:4356-4363) were protected against the development of dengue-4 virusencephalitis when challenged by intracerebral injection. Inoculation ofmice with specific combinations of monoclonal antibodies (Mabs) directedagainst dengue-2 NS1 (Henchal, E. A. et al. 1988 J Gen Virol69:2101-2107) also protects against lethal virus encephalitis uponintracerebral dengue-2 challenge. Other nonstructural proteins are alsoimmunogenic and may participate in eliciting protection (Brinton, M. A.et al. 1998 Clin Diagn Virol 10:129-39).

Herein we have reported the successful construction of several denguevirus replicons which replicate intracellularly without the pre-M and Eproteins required to form infectious virions, including replicons whichcan be expressed from transfected DNA. Towards the goal of devising a“live” dual vaccine based on only non-structural dengue proteins andheterologous HIV material, we report herein that these replicons can beharnessed to express heterologous genes, including HIV gp160 and gp120.Upon introduction into a host's cells, these sub-genomic fragmentsshould replicate intracellularly and support prolonged expression ofdengue and heterologous immunogens without producing the deleted denguestructural proteins and without forming infectious virions.

Development of Dengue Virus Replicons Expressing HIV-1 gp120 and OtherHeterologous Genes: A Tool for Dual Vaccination against Dengue Virus andHIV (Preface)

Toward the goals of providing an additional vector to add to thearmamentarium available to HIV vaccinologists and of creating a bivalentvaccine effective against dengue virus and HIV, we have attempted tocreate vectors which express dengue virus non-structural proteins andHIV immunogens. Herein we have reported the successful construction ofdengue virus replicons which lack structural genes necessary for virionrelease and spreading infection in culture but which can replicateintracellularly and abundantly produce dengue non-structural proteins.Herein we have now expressed heterologous genetic material from thesereplicons.

We cloned into a Δpre-M/E dengue virus replicon genes for either greenfluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the abilityof these constructs to express dengue virus proteins as well as theheterologous proteins in tissue culture after transfection of repliconRNA.

Our conclusions indicate that heterologous proteins were readilyexpressed from these constructs. GFP and gp120 demonstrated minimal orno toxicity. Gp160 expressing replicons were found to express proteinsabundantly at 36 hours post transfection, but after 50 hrs. oftransfection, few replicon positive cells could be found despite thepresence of cellular debris positive for replicon proteins. Thissuggested that gp160 expressed from dengue virus replicons isconsiderably more toxic than either GFP or gp120. The successfulexpression of heterologous proteins, including HIV gp120 for longperiods in culture indicates this vector system should be useful as avaccine vector, given appropriate delivery methods.

Development of Dengue Virus Replicons Expressing HIV-1 gp120 and OtherHeterologous Genes: A Tool for Dual Vaccination against Dengue Virus andHIV

In various previous attempts to express heterologous genes in fulllength, wild type dengue virus, we experienced a very poor success rate,despite attempts to clone heterologous material into various positionsof the genome. Our first efforts to determine whether or notheterologous material could be readily expressed in dengue replicons wasto clone the comparatively tractable green fluorescent protein (GFP)into the Δpre-M/E replicon, into the position from which the pre-M and Egenes had been deleted (FIG. 6). GFP was readily visualized in cultures48 hours post transfection with Δpre-M/E-GFP, as seen in FIG. 7.

Encouraged by the success with GFP, we next looked at Δpre-M/E repliconswith HIV-1 env material cloned into the position of the deleted pre-Mand E genes. We analyzed two clones, Δpre-M/E-gp120 and Δpre-M/E-gp160,expressing HIV-1 gp120 and gp 160 respectively (FIG. 6). Expression ofgenes in the Δpre-M/E-gp120 replicon was reproducibly visualized at48-50 hours post transfection (FIGS. 8 and 9), at a level ofapproximately 1% of the cells, but in many experiments, thecorresponding cultures transfected with the gp160 replicon,Δpre-M/E-gp160, either no fluorescence could be visualized, or onlyfluorescent cells with a bizarre morphology (characterized by debrisand/or degenerative appearance) could be visualized (FIG. 10). However,when we harvested cultures earlier, at 36 hours post transfection withΔpre-M/E-gp160, intact, fluorescing cells were readily found, though themorphology still appeared atypical compared to either that of culturestransfected with wild type dengue virus and dengue replicons or theΔpre-M/E-gp120 replicon (see FIG. 11).

To serve as effective vaccines, it is preferable, if not necessary, thatexpression systems be capable of expressing immunogens for longer than acouple of days. Although we knew from previous experiments that denguereplicons could survive for at least 7 days in culture, the limiteddurability of cells transfected with gp160-expressing replicons raisedthe question of whether or not cells transfected with Δpre-M/E-gp120replicons could survive for similarly long times in culture. Whencultures transfected with Δpre-M/E-gp120 were trypsinized and replatedon day 7 post transfection and then analyzed on day 9 post transfection,fluorescent cells were readily visualized (FIG. 12). In comparison tocultures that were not trypsinized on day 7 post transfection however,these cultures had fewer intact fluorescent cells and more debris.Although this suggests that gp120 expression from a dengue repliconstresses cells, we did find fields with adjacent, gp120 positive cells,suggesting that at least one cell division between day 7 and day 9 hadoccurred in a cell successfully transfected with Δpre-M/E-gp120 (FIG.12, right panel). At 9 days of culture post transfection withΔpre-M/E-gp120, only about 0.1% of the cells or less were positive,which represents a considerable decrease from 48 hours posttransfection.

In the experiments described above and in FIGS. 8 through 12, expressionof dengue replicons with heterologous material from HIV was followedeither using anti-HIV sera or anti-dengue sera, depending on theexperiment. To demonstrate that the same cells were expressing bothdengue proteins and HIV proteins, we used a double label technique, withFITC detecting HIV proteins and rhodamine detecting dengue proteins.FIG. 13 demonstrates the concordance of dengue virus protein and gp120expression in cultures 4 days post transfection with Δpre-M/E-gp120(FIG. 13). The more extensive background of auto fluorescenceencountered when visualizing the rhodamine fluorescence makes low levelsof specific rhodamine fluorescence more difficult to discern, butclearly all intact cells positive for HIV are also positive for dengueproteins. The rhodamine-positive spot in the lower left of the panel iscellular debris and is also positive for dengue proteins, but the FITCfluorescence was not well reproduced by digital photography, though itstill may be visualized on certain monitor/computer combinations.Similar results were obtained at 7 days post transfection (FIG. 14).

Our finding that dengue virus replicons can express heterologous genes,including HIV envelope, for prolonged periods of time in cell culturewithout selection represents a significant step in developing a newvector system potentially capable of delivering immunogens to any hostin whose cells the dengue replicons can replicate. Flavivirus repliconshave previously been demonstrated to express heterologous genes for upto 41 days in tissue culture in Kunjin (Khromykn, A. A. and Westaway, E.D. 1997 J Virol 71:1497-1505). However, these experiments were done inthe presence of selection for the heterologous genes cloned into thereplicons. We have demonstrated heterologous gene expression in theabsence of selection for up to at least 9 days post transfection withchimeric dengue replicons. Although we have formally demonstratedexpression, not replication, our previous demonstration of thereplication of dengue replicons lacking heterologous material suggeststhat the replicons described herein, which contain heterologousmaterial, are indeed replicating. Evidence that cells continue toreplicate and express replicon proteins in both daughter cells aftertransfection with these chimeric replicons further supports theimplication of chimeric replicon replication. Ideally, to serve as dualvaccines against dengue as well as against other pathogens, thereplicons should express the dengue NS1 protein (Schlesinger, J. J. etal. 1987 J Gen Virol 68:853-857; Falgout, B. et al. 1990 J Virol64:4356-4363; Henchal, E. A. et al. 1988 J Gen Virol 69:2101-2107;Brinton, M. A. et al. 1998 Clin Diagn Virol 10:129-39). So far, attemptsto visualize NS1 production by Western blots have failed, presumablybecause of the low transfection efficiencies. However, we havepreviously argued that the replication of dengue replicons could nottake place in the absence of the essential non-structural gene, NS1,which implies that NS1 is being made. The frequencies and fluorescenceintensities of replicon positive cells seen in the experiments reportedherein are comparable to those seen for dengue replicons lackingheterologous material, suggesting that replication of the Δpre-M/Ereplicons containing heterologous material is occurring as well. Thefinding of at least one closely apposed pair of cells expressing highlevels of replicon proteins on day 9 post transfection, two days aftertrypsinization and replating (FIG. 12, right panel), not only impliesreplicon replication, but also implies the expression of NS-1 protein aswell. We contemplate the definitive demonstration of effective NS-1production upon studies of the immune response in animals immunized withthese replicons.

Choice of immunogen remains problematic for these vectors. Clearly,HIV-1 gp 160 is too toxic for prolonged expression. Even thegp120-expressing replicon seems mildly toxic in that the frequency ofgp120 positive cells declines with time in culture post transfectionwith Δpre-M/E-gp120. However, as noted above, we have seen at least oneinstance of putative cellular division at least 7 days after beingsuccessfully transfected by Δpre-M/E-gp120 (FIG. 12, right panel).Experiments are contemplated to determine the feasibility of long termexpression of other HIV-1 immunogens, including gag and tat.

In conclusion, demonstration of long term protein expression by agp120-expressing replicon alone, of course, does not demonstrate thatthe chimeric dengue replicons constitute an effective vaccine. However,at the very least they add to the potential armamentarium available tothe vaccinologist. It is highly likely that a successful HIV vaccinationprotocol will involve multiple immunogens and delivery protocols. Forinstance, mice immunized with attenuated Friend leukemia virus (FLV)develop an immune response whose efficacy is dependent on the additiveeffects of at least three separable spleen cell populations (Dittmer, U.et al. 1999 J Virol 73:3753-3757). By analogy, it may be necessary todevise multiple strategies to obtain a similarly complex and effectiveimmune response in humans against HIV. In animal models of HIV,different immunogens and modes of immunization can induce differentmodes of protection with varying degrees of effectiveness (Heeney, J. etal. 1999 Immunol Lett 66:189-95; Hirsch, V. et al. 1996 J Virol70:3741-3752; Quesada-Rolander, M. et al. 1996 AIDS Res Hum Retroviruses12:993-999; Letvin, N. L. et al 1997 PNAS USA 94:9378-383; Miller, C. J.et al. 1997 J Virol 71:1911-1921; Shibata, R. et al. 1997 J Virol71:8141-8148; Gundlach, B. et al. 1998 J Virol 72:7846-7851). Harnessingmultiple immune responses may be the answer to designing an effectiveHIV vaccine (Nathanson, N. and Mathieson, B. J. 2000 J Infect Dis182:579-589) and the availability of multiple vectors should facilitatethe harnessing of multiple responses.

Culturing of Dengue Virus

Dengue virus strains DEN1/WP and DEN2/NGC, kindly provided by Dr. LewisMarkoff, (Polo, S. et al. 1997 J Virol 71:5366-5374; Pur, B. et al. 2000Virus Genes 20:57-63) were passaged in monkey LLC-MK2 cells at 37° C. ina humidified incubator under 5% CO₂, using Medium 199 plus 10% fetalbovine serum (FBS) and 50 μg of Gentamicin per ml. The cells weretrypsinized a day before virus infection and plated to reachapproximately 80% confluence on the day of infection. Infections weretypically at an MOI of 0.01 PFU/cell in Medium 199 plus 2% FBS.

In Vitro Mutagenesis

Heterologous genes were cloned into the previously described Δpre-M/Ereplicon, into the position previously occupied by the pre-M/E genes.DNA fragments used for desired regions of heterologous genes (see FIG.6) were synthesized by polymerase chain reaction (PCR) from shortoverlapping primers. For the Green Fluorescent Protein (GFP) gene, the5′ primer was 5′CGAAAAAAGGCGAGAAATACGCCTTTCAATATGCTGAAACGCGAGAGAATGGTGAGCAAGGGCGAGGAGCTG3′ (SEQ ID. NO.: 26) and the 3′ primerwas 5′AAGGTCAAAATTCAACAGCTGCTTGTACAGCTCGTCCATGCC3′ (SEQ ID. NO.: 27).For HIV-1 gp120 gene, the 5′ primer was 5′ATCATTATGCTGAATCCAACAGTGATGGCGTTCCATTTACCACACGTAACATGAGAGTGATGGGGATCA GGAAG3′ (SEQ ID.NO.: 28) and the 3′ primer was 5′AAGGTCAAAATTCAACAGCTGGGTGGGTGCTAATCCTAATGGTTC3′ (SEQ ID. NO.: 29). GFP and HIV-1 gp120 werefused with FMDV/2A self cleaving protein sequence to replace naturalcleavage sites in the dengue polyprotein. These sites seem to loosetheir activity when juxtaposed with heterologous material. PCR was usedto amplify DNA coding for the FMDV/2A self cleaving protein. The 5′primer was 5′CAGCTGTTGAATTTTGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGCCCC′ (SEQ ID. NO.: 30) and the 3′ primer was

(SEQ ID. NO.: 31) 5′ATACAGCGTCACGACTCCCACCAATACTAGTGACACAGACAGTGAGGTGCTGGGGCCAGGGTTGGACTCGAC3′.

Dengue-2 virus cDNA cloned in the yeast shuttle vector pRS424,linearized by excision of a short Bam H1 fragment was transfected intocompetent (Spencer, F. et al. 1993 Methods Companion Methods Enzymol5:161-175) S. cerevisiae YPH857, kindly provided by Barry Falgout(CBER/FDA), along with the appropriate PCR fragment spanning the desireddeletion. Yeast colonies which grew on tryptophan minus platesrepresented vectors which had recircularized by homologous recombinationwith these PCR fragments (Hirsch, V. et al. 1996 J Virol 70:3741-3752).DNA from these colonies was transformed into E. coli STBL 2 cells (LifeTechnologies, Inc.) to make sufficient quantities of dengue recombinant,genomic-length DNAs for characterization and analysis.

Expression of Virus and Replicons in Cells

The full length virus and replicon cDNA plasmids isolated from STBL 2cells were linearized with SacI, purified by Qiagen chromatography, andeluted by RNAase-free water in preparation for transcription. Thetranscription reaction mixtures contained 1 μg of linearized DNA; 0.5 mM(each) ATP, CTP, and UTP; 0.1 mM GTP; 0.5 mM cap analog (NEBL); 10 mMDTT; 40 U of Rnasin (Promega); 30 U of SP6 RNA polymerase; and 1×SP6 RNApolymerase buffer (Promega) in a volume of 30 μl. The reaction mixtureswere incubated at 40° C. for 2 hr. Aliquots (12.5 μl) of the reactionmixtures, containing full length viral RNA, were used to transfectapproximately 2×10⁶ Monkey LLC-MK2 cells in phosphate-buffered saline(PBS) by electroporation in a 0.4 cm gap electroporation cuvette. Eachcuvette was pulsed at 200 V, 950 μF using a BioRad Genepulselectroporator. The cells were then resuspended in growth medium andplated on the appropriate tissue culture dish.

After electroporation, cells were either plated directly on multiwellplates for harvest at short time periods (typically 4 days or less) oron tissue culture dishes for trypsinization and seeding onto multiwellplates one or two days before final harvest for longer time periods.

Immuno-Histochemical Methods

For immunofluorescent detection of dengue-specific proteins, cellsgrowing on chamber slides were rinsed in room-temperature PBS and thenfixed in cold acetone for 10 min at −20° C. After being air dried, eachchamber was covered with 50 μl of a 1:50 dilution of DEN2-specifichyperimmune mouse ascitic fluid (HMAF, American Type Culture Collection)in PBS plus 2% normal goat serum and incubated at room temperature for 1h in a humidified atmosphere and then rinsed twice in PBS. Afterwashing, cells were subsequently incubated with a 1:100 dilution offluorescein isothiocyanate-labeled goat anti-mouse antibodies(Kirkegaard and Perry Laboratory) and rinsed twice in PBS. For detectionof HIV-specific proteins, the same protocol was used except that cellswere initially incubated with human HIV-1 serum from WaldheimPharmazeutika Ges.m.b.H. Neufeld-Vienna, Austria and then subsequentlyincubated with fluorescent-labeled goat anti-human antibody. Cells insome, but not all experiments were counterstained with 0.02% Evans Blue.

For dual labeling, the first antibodies were a 1:50 dilution of denguetype 2 specific hyperimmune mouse ascitic fluid (HMAF, American typeculture collection) and a 1:100 dilution of human HIV positive serum inPBS plus 2% normal goat serum. The second antibodies were a 1:100dilution of FITC-labeled goat anti-human antibodies (WaldeimPharmazeutika) and a 1:50 dilution of goat anti-mouse IgG-L-Rhodamine(Boehringer Mannheim Biochemicals).

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All patents, patentapplications and publications referred to above are hereby incorporatedby reference.

1. A subgenomic replicon of dengue virus origin comprising a deletionfor the sequence coding for E structural protein (ΔE).
 2. The subgenomicreplicon of dengue virus origin of claim 1 further comprising adeletion-for the sequence coding for PreM (ΔME).
 3. The subgenomicreplicon of dengue virus origin of claim 2 further comprising a deletionfor the sequence coding for C (ΔCME).
 4. The subgenomic replicon ofclaim 1 wherein the dengue virus origin is dengue virus type 1, 2, 3 or4 origin.
 5. The subgenomic replicon of claim 2 wherein the dengue virusorigin is dengue virus type 1, 2, 3 or 4 origin.
 6. The subgenomicreplicon of claim 3 wherein the dengue virus origin is dengue virus type1, 2, 3 or 4 origin.
 7. The subgenomic replicon of dengue virus originof claim 1 comprising a deletion for the sequence coding for C, PreM,and E structural proteins (ΔCME), for PreM and E structural proteins(ΔME), or for E structural protein (ΔE); and further comprising part orall of the 5′UTR; at least about the first 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, or 175 nucleotides of C protein; at least aboutthe last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or 175nucleotides of E protein; substantially all of the nonstructural region;and part or all of the 3′UTR.
 8. The subgenomic replicon of dengue virusorigin of claim 4 comprising a deletion for the sequence coding for C,PreM, and E structural proteins (ΔCME), for PreM and E structuralproteins (ΔME), or for E structural protein (ΔE); and further comprisingpart or all of the 5′UTR; at least about the first 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, or 175 nucleotides of C protein; at leastabout the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or175 nucleotides of E protein; substantially all of the nonstructuralregion; and part or all of the 3′UTR.
 9. The subgenomic replicon ofdengue virus origin of claim 1 which is adapted to receive at least anucleotide sequence without disrupting its replication capabilities. 10.A vaccine comprising the subgenomic replicon of dengue virus origin ofclaim 1, optionally which is adapted to receive at least a nucleotidesequence without disrupting its replication capabilities, and apharmaceutically acceptable carrier.
 11. A therapeutic comprising thesubgenomic replicon of dengue virus origin of claim 9 and apharmaceutically acceptable carrier.
 12. A dengue virus like particlecomprising a subgenomic replicon of dengue virus origin of claim 9further comprising structural proteins of the homologous dengue viruswherein said structural proteins encapsulate said subgenomic replicon.13. A method of immunization comprising administering to an individualin need thereof a subgenomic replicon of dengue virus origin of claim 9.14. A method of immunization comprising administering to an individualin need thereof a dengue virus like particle of claim
 12. 15. A methodof treatment comprising administering to an individual in need thereofthe subgenomic replicon of dengue virus origin of claim
 9. 16. A methodof treatment comprising administering to an individual in need thereof adengue virus like particle of claim 12.